Inhibition of NF-jB by a cell permeable form of IjBa induces apoptosis in eosinophils Satoko Fujihara a, * , Ellis Jaffray b , Stuart N. Farrow c , Adriano G. Rossi a , Christopher Haslett a , Ronald T. Hay b a Department of Medicine, Centre for Inflammation Research, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, UK b Centre for Biomolecular Sciences, School of Biology, University of St. Andrews, North Haugh, St. Andrews, Fife KY16 9ST, UK c The Asthma Biology Department, GlaxoSmithKline, Stevenage, Herts SG1 2NT, UK Received 15 November 2004 Available online 7 December 2004 Abstract An 11 amino acid HIV-TAT peptide can deliver target proteins into a variety of cells in a receptor-independent manner. To gen- erate a highly specific inhibitor of the transcription factor NF-jB, we have fused the TAT-peptide to a version of IjBa that is resis- tant to signal-induced degradation. TAT-IjBa(S32A, S36A) inhibited NF-jB-dependent transcription in HeLa and A549 cells by retaining NF-jB p65 in the cytoplasm. Introduction of TAT-IjBa(S32A, S36A) into human eosinophils inhibited the nuclear trans- location of NF-jB and induced apoptosis. Thus, continuous NF-jB-dependent transcription is important for eosinophil survival. While eosinophils are normally refractive to standard methods of gene delivery, the ability of TAT fusion proteins to be taken up by these cells should enable a detailed molecular analysis of survival pathways in these cells. Ó 2004 Elsevier Inc. All rights reserved. Keywords: HIV-TAT; NF-jB; IjBa; TAT-IjBa; Eosinophils; Apoptosis Excessive eosinophil recruitment and activation at inflammatory sites are likely to cause surrounding tissue damage by liberation of their toxic granule contents. Toxic granule proteins are important for defence against parasites, but if they are released in the host tissue when eosinophils are inappropriately activated, they will con- tribute to the progression of inflammatory diseases such as asthma. Thus, levels of eosinophilic toxic granule components such as major basic protein, eosinophil cat- ionic protein, eosinophil peroxidase, and cysteinyl leu- kotrienes have been found in significantly increased amounts in the airways of asthmatic patients and animal models, suggesting that they may be responsible for the airway tissue damage and remodelling [1–3]. An additional function of eosinophils is the production of pro-inflammatory cytokines including IL-1, IL-4, IL-6, IL-8, TNFa, GM-CSF, and IL-5 [4]. Synthesis of these proteins in eosinophils is dependent on the activation of intracellular signalling cascades and key transcription factors. Some of these proteins trigger the activation of inflammatory cells, including eosinophils, thereby con- tributing to an amplification of the inflammatory re- sponse and disease progression. Therefore, effective removal of eosinophils from inflammatory sites may be a key factor for successful resolution of the inflamma- tory process. As previously shown from our study [5,6] and others [7,8] NF-jB is a key regulator of eosinophil activation and survival. NF-jB is a family of transcription factors that regu- lates survival and inflammatory responses [9,10]. The NF-jB family contains the proteins p65/Rel A, c-Rel, 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.11.090 * Corresponding author. Present address: Medicine and Therapeu- tic, University of Dundee, Ninewells Hospital, Dundee, Scotland UKDD1 9SY, UK. Fax: +44 1382 64972. E-mail address: s.fujihara@dundee.ac.uk (S. Fujihara). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 326 (2005) 632–637 BBRC